Methods and compounds for inhibiting mrp1

ABSTRACT

The present invention further relates to a method of inhibiting MRP1 in a mammal which comprises administering to a mammal in need thereof an effective amount of a compound of formula (I).

[0001] Along with surgery and radiotherapy, chemotherapy continues to bean effective therapy for many cancers. In fact, several types of cancer,such as Hodgkin's disease, large cell lymphoma, acute lymphocyticleukemia, testicular cancer and early stage breast cancer, are nowconsidered to be curable by chemotherapy. Other cancers, such as ovariancancer, small cell lung and advanced breast cancer, while not yetcurable, are exhibiting positive response to combination chemotherapy.

[0002] One of the most important unsolved problems in cancer treatmentis drug resistance. After selection for resistance to a single cytotoxicdrug, cells may become cross-resistant to a whole range of drugs withdifferent structures and cellular targets, e.g., alkylating agents,antimetabolites, hormones, platinum-containing drugs, and naturalproducts. This phenomenon is known as multidrug resistance (MDR). Insome types of cells this resistance is inherent, while in others, suchas small cell lung cancer, it is usually acquired. Such resistance isknown to be multifactorial and is conferred by at least two proteins:the 170 kDa P-glycoprotein (MDR1) and the more recently identified 190kDa multidrug resistance protein (MRP1). Although both MDR1 and MRP1belong to the ATP-binding cassette superfamily of transport proteins,they are structurally very different molecules and share less than 15%amino acid homology.

[0003] Despite the structural divergence between the two proteins, by1994 there were no known consistent differences in the resistancepatterns of MDR1 and MRP1 cell lines. However, the association, or lackthereof, of MRP1 and resistance to particular oncolytics is known. SeeCole, et. al., “Pharmacological Characterization of Multidrug ResistantMRP-transfected Human Tumor Cells”, Cancer Research, 54:5902-5910, 1994.Doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide aresubstrates of MRP1, i.e., MRP1 can bind to these oncolytics andredistribute them away from their site of action, the nucleus, and outof the cell. Id. and Marquardt, D., and Center, M. S., Cancer Research,52:3157, 1992.

[0004] Doxorubicin, daunorubicin, and epirubicin are members of theanthracycline class of oncolytics. They are isolates of various strainsof Streptomyces and act by inhibiting nucleic acid synthesis. Theseagents are useful in treating neoplasms of the bone, ovaries, bladder,thyroid, and especially the breast. They are also useful in thetreatment of acute lymphoblastic and myeloblastic leukemia, Wilm'stumor, neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin'slymphomas, and bronchogenic carcinoma.

[0005] Vincristine, a member of the vinca alkaloid class of oncolytics,is an isolate of a common flowering herb, the periwinkle plant (Vincarosea Linn). The mechanism of action of vincristine is still underinvestigation but has been related to the inhibition of microtubuleformation in the mitotic spindle. Vincristine is useful in the treatmentof acute leukemia, Hodgkin's disease, non-Hodgkin's malignant lymphomas,rhabdomyosarcoma, neuroblastoma, and Wilm's tumor.

[0006] Etoposide, a member of the epipodophyllotoxin class ofoncolytics, is a semisynthetic derivative of podophyllotoxin. Etoposideacts as a topoisomerase inhibitor and is useful in the therapy ofneoplasms of the testis, and lung.

[0007] Additionally, PCT publications WO 99/51236, WO 99/51228, and WO99/51227 disclose certain compounds known to be inhibitors of MRP1.

[0008] It is presently unknown what determines whether a cell line willacquire resistance via a MDR1 or MRP1 mechanism. Due to the tissuespecificity of these transporters and/or in the case where one mechanismpredominates or is exclusive, it would be useful to have a selectiveinhibitor of that one over the other. Furthermore, when administering adrug or drugs that are substrates of either protein, it would beparticularly advantageous to coadminister an agent that is a selectiveinhibitor of that protein. It is, therefore, desirable to providecompounds that are selective inhibitors of MDR1 or MRP1.

[0009] The present invention relates to a compound of formula I:

[0010] where:

[0011] E is a bond or —C(R⁴)(R⁴)—;

[0012] R¹ is independently at each occurrence hydrogen or C₁-C₆ alkyl;

[0013] R² is independently at each occurrence hydrogen or C₁-C₆ alkyl;

[0014] R³ is independently at each occurrence hydrogen, C₁-C₆ alkyl,optionally substituted C₃-C₈ cycloalkyl, optionally substituted (C₁-C₄alkyl) C₃-C₈ cycloalkyl, optionally substituted (C₁-C₄ alkyl)-aryl,optionally substituted aryl, optionally substituted (C₁-C₄alkyl)-heterocycle, optionally substituted heterocycle, C₁-C₆ alkoxy,optionally substituted O—(C₃-C₈ cycloalkyl), optionally substituted(C₁-C₄ alkoxy) C₃-C₈ cycloalkyl, optionally substituted (C₁-C₄alkoxy)-aryl, optionally substituted O-aryl, optionally substituted(C₁-C₄ alkoxy)-heterocycle, or optionally substituted O-heterocycle;

[0015] R⁴ is independently at each occurrence hydrogen or C₁-C₆ alkyl;

[0016] R⁵ is independently at each occurrence hydrogen or C₁-C₆ alkyl;

[0017] or a pharmaceutical salt thereof.

[0018] The present invention further relates to a method of inhibitingMRP1 in a mammal which comprises administering to a mammal in needthereof an effective amount of a compound of formula I.

[0019] In another embodiment, the present invention relates to a methodof inhibiting a resistant neoplasm, or a neoplasm susceptible toresistance in a mammal which comprises administering to a mammal in needthereof an effective amount of a compound of formula I in combinationwith an effective amount of an oncolytic agent.

[0020] The present invention also relates to a pharmaceuticalformulation comprising a compound of formula I in combination with oneor more oncolytics, pharmaceutical carriers, diluents, or excipientstherefor.

[0021] Additionally, the present invention relates to a pharmaceuticalformulation comprising a compound of formula I.

[0022] The current invention concerns the discovery that compounds offormula I are selective inhibitors of multidrug resistant protein(MRP1), and are thus useful in treating MRP1 conferred multidrugresistance (MDR) in a resistant neoplasm and a neoplasm susceptible toresistance.

[0023] The terms “inhibit” as it relates to MRP1 and “inhibiting MRPl”refer to prohibiting, alleviating, ameliorating, halting, restraining,slowing or reversing the progression of, or reducing MRP1's ability toredistribute an oncolytic away from the oncolytic's site of action, mostoften the neoplasm's nucleus, and out of the cell.

[0024] As used herein, the term “effective amount of a compound offormula I” refers to an amount of a compound of the present inventionwhich is capable of inhibiting MRP1. The term “effective amount of anoncolytic agent” refers to an amount of oncolytic agent capable ofinhibiting a neoplasm, resistant or otherwise.

[0025] The term “inhibiting a resistant neoplasm, or a neoplasmsusceptible to resistance” refers to prohibiting, halting, restraining,slowing or reversing the progression of, reducing the growth of, orkilling resistant neoplasms and/or neoplasms susceptible to resistance.

[0026] The term “resistant neoplasm” refers to a neoplasm, which isresistant to chemotherapy where that resistance is conferred in part, orin total, by MRP1. Such neoplasms include, but are not limited to,neoplasms of the bladder, bone, breast, lung (small-cell), testis, andthyroid and also includes more particular types of cancer such as, butnot limited to, acute lymphoblastic and myeloblastic leukemia, Wilm'stumor, neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin'slymphomas, and bronchogenic carcinoma.

[0027] A neoplasm, which is “susceptible to resistance”, is a neoplasmwhere resistance is not inherent nor currently present but can beconferred by MRP1 after chemotherapy begins. Thus, the methods of thisinvention encompass a prophylactic and therapeutic administration of acompound of formula I.

[0028] The term “chemotherapy” refers to the use of one or moreoncolytic agents where at least one oncolytic agent is a substrate ofMRP1. A “substrate of MRP1” is an oncolytic that binds to MRP1 and isredistributed away from the oncolytics site of action (the nucleus ofthe neoplasm) and out of the cell, thus, rendering the therapy lesseffective. Preferred oncolytic agents are doxorubicin, daunorubicin,epirubicin, vincristine, and etoposide.

[0029] The terms “treat” or “treating” bear their usual meaning whichincludes preventing, prohibiting, alleviating, ameliorating, halting,restraining, slowing or reversing the progression, or reducing theseverity of MRP1 derived drug resistance in a multidrug resistant tumor.

[0030] In the general formulae of the present document, the generalchemical terms have their usual meanings. For example, the term “C₁-C₄alkyl” refers to methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl,cyclobutyl, s-butyl, and t-butyl. The term “C₁-C₆ alkyl” refers to amonovalent, straight or branched saturated hydrocarbon containing from 1to 6 carbon atoms. Additionally, the term “C₁-C₆ alkyl” includes C₁-C₄alkyl groups and C₃-C₆ cycloalkyls. The term “C₁-C₆ alkyl” includes, butis not limited to, cyclopentyl, pentyl, hexyl, cyclohexyl, and the like.The term “C₃-C₈ cycloalkyl” refers to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The term “C₅-C₇cycloalkyl” refers to cyclopentyl, cyclohexyl, and cycloheptyl. The term“C₆-C₁₀ bicycloalkyl” refers to bicyclo-[2.1.1]hexanyl, [2.2.1]heptanyl,[3.2.1]octanyl, [2.2.2]octanyl, [3.2.2]nonanyl, [3.3.1]nonanyl,[3.3.2]decanyl, and [4.3.1]decanyl ring systems.

[0031] The terms “C₁-C₄ alkoxy” and “C₁-C₆ alkoxy” refer to moieties ofthe formula O—(C₁-C₄ alkyl) and O—(C₁-C₆ alkyl) respectively.

[0032] The term “optionally substituted C₃-C₈ cycloalkyl” refers to aC₃-C₈ cycloalkyl unsubstituted or substituted once with a phenyl,substituted phenyl, or CO₂R¹ group.

[0033] The terms “optionally substituted (C₁-C₄ alkyl)-(C₃-C₈cycloalkyl)” refers to optionally substituted C₃-C₈ cycloalkyl linkedthrough a C₁-C₄ alkyl, optionally substituted with halo or hydroxy.

[0034] The term “optionally substituted C₆-C₁₀ bicycloalkyl” refers to aC₆-C₁₀ bicycloalkyl unsubstituted or substituted once with a phenyl,substituted phenyl, or CO₂R¹ group.

[0035] The term “halo” or “halide” refers to fluoro, chloro, bromo, andiodo.

[0036] The term “aryl” refers to phenyl and naphthyl.

[0037] The terms “optionally substituted aryl” refers to a phenyl andnaphthyl group respectively optionally substituted from 1 to 5 timesindependently with C₁-C₆ alkyl, C₁-C₄ alkoxy, halo, hydroxy,trifluoromethyl, NR⁴R⁵, SO₂NR⁴R⁵, NH-Pg, C₁-C₆ alkoxy, benzyloxy,C(O)R⁴, C₅-C₇ cycloalkyl, trifluoromethoxy, SR¹, cyano, or nitro.

[0038] The terms “optionally substituted (C₁-C₄ alkyl)-aryl” refers tooptionally substituted aryl linked through a C₁-C₄ alkyl, optionallysubstituted with halo, trifluoromethyl, or hydroxy.

[0039] The term “heterocycle” is taken to mean stable unsaturated andsaturated 3 to 6 membered rings containing from 1 to 4 heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, saidrings being optionally benzofused. All of these rings may be substitutedwith up to three substituents independently selected from the groupconsisting of halo, C₁-C₄ alkoxy, C₁-C₄ alkyl, cyano, nitro, hydroxy,—S(O)_(m)—(C₁-C₄ alkyl) and —S(O)_(m)-phenyl where m is 0, 1 or 2.Saturated rings include, for example, pyrrolidinyl, piperidinyl,piperazinyl, tetrahydrofuryl, oxazolidinyl, morpholino, dioxanyl,pyranyl, and the like. Benzofused saturated rings include indolinyl,1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl and thelike. Unsaturated rings include furyl, thienyl, pyridinyl, pyrrolyl,N-methylpyrrolyl, oxazolyl, isoxazolyl, pyrazolyl, imidazolyl,triazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, pyrimidinyl, pyrazinyl,pyridazinyl, and the like. Benzofused unsaturated rings includeisoquinolinyl, benzoxazolyl, benzthiazolyl, quinolinyl, benzofuranyl,thionaphthyl, indolyl and the like.

[0040] The term “substituted heterocycle” refers to a heterocyclic ringsubstituted 1 or 3 times independently with a C₁-C₆ alkyl, halo, benzyl,phenyl, trifluoromethyl. Saturated heterocyclic rings may beadditionally substituted 1 or 2 times with an oxo group, however, totalsubstitution of the saturated heterocyclic ring may not exceed twosubstituents.

[0041] The terms “optionally substituted (C₁-C₄ alkyl)-heterocycle”refers to optionally substituted heterocycle linked through a C₁-C₄alkyl, optionally substituted with halo or hydroxy.

[0042] The term “amino acid ester” as used in this specification refersto an amino acid where the carboxy group is substituted with a C₁-C₆alkyl or benzyl group. That is, the alkyl group when taken together withthe carboxy group forms a C₁-C₆ alkyl ester. A skilled artisan wouldappreciate that some amino acids have two carboxy groups that may besubstituted with a C₁-C₆ alkyl group, for example, aspartic acid andglutamic acid. This invention contemplates the possibility of amino acidmono- or diesters in these circumstances.

[0043] The term “protecting group” (Pg) refers to an amino protectinggroup or a hydroxy protecting group. The species of protecting groupwill be evident from whether the “Pg” group is attached to a nitrogenatom (amino protecting group) or attached to an oxygen atom (hydroxyprotecting group).

[0044] The term “amino protecting group” as used in this specificationrefers to a substituent(s) of the amino group commonly employed to blockor protect the amino functionality while reacting other functionalgroups on the compound. Examples of such amino-protecting groups includethe formyl group, the trityl group, the phthalimido group, the acetylgroup, the trichloroacetyl group, the chloroacetyl, bromoacetyl, andiodoacetyl groups, urethane-type blocking groups such asbenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (“FMOC”), and the like;and like amino protecting groups. The species of amino protecting groupemployed is not critical so long as the derivatized amino group isstable to the condition of subsequent reaction(s) on other positions ofthe molecule and can be removed at the appropriate point withoutdisrupting the remainder of the molecule. Similar amino protectinggroups used in the cephalosporin, penicillin, and peptide arts are alsoembraced by the above terms. Further examples of groups referred to bythe above terms are described by T. W. Greene, “Protective Groups inOrganic Synthesis”, John Wiley and Sons, New York, N.Y., 1991, Chapter 7hereafter referred to as “Greene”. A preferred amino protecting group ist-butyloxycarbonyl.

[0045] The term “hydroxy protecting group” denotes a group understood byone skilled in the organic chemical arts of the type described inChapter 2 of Greene. Representative hydroxy protecting groups include,for example, ether groups including methyl and substituted methyl ethergroups such as methyl ether, methoxymethyl ether, methylthiomethylether, tert-buylthiomethyl ether, (phenyldimethylsilyl)methoxy-methylether, benzyloxymethyl ether, p-methoxybenzyloxy-methyl ether, andtert-butoxymethyl ether; substituted ethyl ether groups such asethoxyethyl ether, 1-(2-chloroethoxy)-ethyl ether,2,2,2-trichloroethoxymethyl ether, and 2-(trimethylsilyl)ethyl ether;isopropyl ether groups; phenyl and substituted phenyl ether groups suchas phenyl ether, p-chlorophenyl ether, p-methoxyphenyl ether, and2,4-dinitrophenyl ether; benzyl and substituted benzyl ether groups suchas benzyl ether, p-methoxybenzyl ether, o-nitrobenzyl ether, and2,6-dichlorobenzyl ether; and alkylsilyl ether groups such as trimethyl-triethyl- and triisopropylsilyl ethers, mixed alkylsilyl ether groupssuch as dimethylisopropylsilyl ether, and diethylisopropylsilyl ether;and ester protecting groups such as formate ester, benzylformate ester,mono-, di-, and trichloroacetate esters, phenoxyacetate ester, andp-chlorophenoxyacetate and the like. The species of hydroxy protectinggroup employed is not critical so long as the derivatized hydroxy groupis stable to the conditions of subsequent reaction(s) on other positionsof the intermediate molecule and can be selectively removed at theappropriate point without disrupting the remainder of the moleculeincluding any other hydroxy protecting group(s).

[0046] The term “carbonyl activating group” refers to a substituent of acarbonyl that increases the susceptibility of that carbonyl tonucleophilic addition. Such groups include, but are not limited to,alkoxy, aryloxy, nitrogen containing unsaturated heterocycles, or aminogroups such as oxybenzotriazole, imidazolyl, nitrophenoxy,pentachloro-phenoxy, N-oxysuccinimide, N,N′-dicyclohexylisoure-O-yl,N-hydroxy-N-methoxyamino, and the like; acetates, formates, sulfonatessuch as methanesulfonate, ethanesulfonate, benzenesulfonate, orp-toluenylsulfonate, and the like; and halides especially chloride,bromide, or iodide.

[0047] The term “carbonyl activating reagent” refers to a reagent thatconverts the carbonyl of a carboxylic acid group to one that is moreprone to nucleophilic addition and includes, but is not limited to, suchreagents as those found in “The Peptides”, Gross and Meienhofer, Eds.,Academic Press (1979), Ch. 2 and M. Bodanszky, “Principles of PeptideSynthesis”, 2^(nd) Ed., Springer-Verlag Berlin Heidelberg, 1993,hereafter referred to as “The Peptides” and “Peptide Synthesis”respectively. Specifically, carbonyl activating reagents include thionylbromide, thionyl chloride, oxalyl chloride, and the like; alcohols suchas nitrophenol, pentachlorophenol, and the like; amines such asN-hydroxy-N-methoxyamine and the like; acid halides such as acetic,formic, methanesulfonic, ethanesulfonic, benzenesulfonic, orp-tolylsulfonic acid halide, and the like; and compounds such as1,1′-carbonyldiimidazole, benzotriazole, imidazole,N-hydroxysuccinimide, dicyclohexylcarbodiimide, and the like.

[0048] In general, the term “pharmaceutical” when used as an adjectivemeans substantially non-toxic to living organisms. For example, the term“pharmaceutical salt” as used herein, refers to salts of the compoundsof formula I which are substantially non-toxic to living organisms. See,e.g., Berge, S. M, Bighley, L. D., and Monkhouse, D. C., “PharmaceuticalSalts”, J. Pharm. Sci., 66:1, 1977. Typical pharmaceutical salts includethose salts prepared by reaction of the compounds of formula I with aninorganic or organic acid or base. Such salts are known as acid additionor base addition salts respectively. These pharmaceutical saltsfrequently have enhanced solubility characteristics compared to thecompound from which they are derived, and thus are often more amenableto formulation as liquids or emulsions.

[0049] The term “acid addition salt” refers to a salt of a compound offormula I prepared by reaction of a compound of formula I with a mineralor organic acid. For exemplification of pharmaceutical acid additionsalts see, e.g., Berge, S. M, Bighley, L. D., and Monkhouse, D. C., J.Pharm. Sci., 66:1, 1977. Since compounds of this invention can be basicin nature, they accordingly react with any of a number of inorganic andorganic acids to form pharmaceutical acid addition salts.

[0050] The pharmaceutical acid addition salts of the invention aretypically formed by reacting the compound of formula I with an equimolaror excess amount of acid. The reactants are generally combined in amutual solvent such as diethylether, tetrahydrofuran, methanol, ethanol,isopropanol, benzene, and the like. The salts normally precipitate outof solution within about one hour to about ten days and can be isolatedby filtration or other conventional methods.

[0051] Acids commonly employed to form acid addition salts are inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric acid, and the like, and acids commonlyemployed to form such salts are inorganic acids such as hydrochloricacid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid,and the like, and organic acids, such as p-toluenesulfonic acid,methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, acetic acid and thelike. Examples of such pharmaceutically acceptable salts thus are thesulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,β-hydroxybutyrate, glycollate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate and the like.

[0052] The term “base addition salt” refers to a salt of a compound offormula I prepared by reaction of a compound of formula I with a mineralor organic base. For exemplification of pharmaceutical base additionsalts see, e.g., Berge, S. M, Bighley, L. D., and Monkhouse, D. C., J.Pharm. Sci., 66:1, 1977. This invention also contemplates pharmaceuticalbase addition salts of compounds of formula I. The skilled artisan wouldappreciate that some compounds of formula I may be acidic in nature andaccordingly react with any of a number of inorganic and organic bases toform pharmaceutical base addition salts. Examples of pharmaceutical baseaddition salts are the ammonium, lithium, potassium, sodium, calcium,magnesium, methylamino, diethylamino, ethylene diamino, cyclohexylamino,and ethanolamino salts, and the like of a compound of formula I.

[0053] While all of the compounds of the present invention are useful,certain of the compounds are particularly interesting and are preferred.The following listing sets out several groups of preferred compounds. Itwill be understood that each of the listings may be combined with otherlistings to create additional groups of preferred embodiments.

[0054] 1) E is a bond;

[0055] 2) E is —C(R⁴)(R⁴)—;

[0056] 3) When E is —C(R⁴)(R⁴)—, R⁴ is hydrogen;

[0057] 4) When E is —C(R⁴)(R⁴)—, R⁴ is methyl;

[0058] 5) R¹ and R² are hydrogen;

[0059] 6) R³ is C₁-C₆ alkyl;

[0060] 7) R³ is optionally substituted C₃-C₈ cycloalkyl;

[0061] 8) R³ is optionally substituted (C₁-C₄ alkyl) C₃-C₈ cycloalkyl;

[0062] 9) R³ is optionally substituted (C₁-C₄ alkyl)-aryl;

[0063] 10) R³ is optionally substituted aryl;

[0064] 11) R³ is optionally substituted (C₁-C₄ alkyl)-heterocycle;

[0065] 12) R³ is optionally substituted heterocycle, C₁-C₆ alkoxy;

[0066] 13) R³ is optionally substituted O—(C₃-C₈ cycloalkyl);

[0067] 14) R³ is optionally substituted (C₁-C₄ alkoxy) C₃-C₈ cycloalkyl;

[0068] 15) R³ is optionally substituted (C₁-C₄ alkoxy)-aryl;

[0069] 16) R³ is optionally substituted O-aryl;

[0070] 17) R³ is optionally substituted (C₁-C₄ alkoxy)-heterocycle;

[0071] 18) R³ is optionally substituted O-heterocycle;

[0072] 19) R⁵ is hydrogen;

[0073] 20) R⁵ is methyl;

[0074] 21) The compound is a pharmaceutical salt; and

[0075] 22) The compound is the hydrochloride salt.

[0076] The compounds of the present invention can be prepared by avariety of procedures, some of which are illustrated in the Schemesbelow. The particular order of steps required to produce the compoundsof formula I is dependent upon the particular compound beingsynthesized, the starting compound, and the relative lability of thesubstituted moieties.

[0077] Compounds of formula I may be prepared from compounds of formulaII as illustrated in Scheme 1 below, wherein Y is C(O)R⁴ or-E-NR⁵C(O)R³, R¹ and R² are hydrogen, and E, R³, R⁴, and R⁵ are asdescribed supra.

[0078] Compounds of formula I may be prepared by combining a compound offormula II in a suitable solvent, preferably acetonitrile/water (5:1ratio), and adding a suitable reducing agent, such ashexacarbonylmolybdenum.

[0079] The reactants are typically combined at a temperature from about0° C. to about 100° C. The reactants are preferably combined at roomtemperature and the resulting solution is typically heated to reflux andmixed until the reaction is complete, as measured by the consumption ofthe substrate. The final product may be isolated and/or purified bystandard techniques well known in the art.

[0080] Compounds of formula II may be prepared from compounds of formulaIII as illustrated in Scheme 2 below, wherein Y is C(O)R⁴ or-E-NR⁵C(O)R³.

[0081] Compounds of formula II may be prepared by dissolving orsuspending a compound of formula III in a suitable solvent, preferablydimethylformamide, and adding a suitable base, such as potassiummethoxide, potassium tert-butoxide, potassium carbonate, sodiumhexamethyldisilazane, or preferably potassium hexamethyldisilazane. Thebase is typically employed in an one to one ratio. However, as theskilled artisan would appreciate, a slight molar excess, usually inabout a 1.1 to about a 3 fold molar excess relative to the compound offormula III, is acceptable.

[0082] The reactants are typically combined at a temperature from about0° C. to about 100° C. The reactants are preferably combined at roomtemperature and the resulting solution is typically mixed for from about5 minutes to about 18 hours, preferably from about 15 minutes to about 3hours.

[0083] Any protecting groups remaining in the cyclized compound offormula I may be removed as taught in Greene to provide the compounds offormula II. Preferred choices of protecting groups and methods for theirremoval may be found in the Preparations and Examples sections below.

[0084] Compounds of formula II may be prepared from compounds of formula(i) as illustrated in Scheme 3 below where E, R³, R⁴, and R⁵ are asdescribed supra.

[0085] The compounds of formula (i) may be reductively aminated to formthe compounds of formula II. Reductive aminations are well knowntransformations, see, e.g., Larock, “Comprehensive OrganicTransformations”, pg. 421, VCH Publishers, New York, N.Y., 1989,hereafter referred to as “Larock”.

[0086] Amines of formula (a) may be dissolved or suspended in a suitablesolvent, optionally in the presence of a suitable base, preferablyN-methyl morpholine or triethylamine, when the compound of formula IIIis an acid addition salt to convert the salt to its free amine form, anda compound of formula (i) is added. A Lewis acid catalyst, such astitanium(IV) isopropoxide, may optionally be employed. Once it isdetermined that the compound of formula (i) has been substantiallyconsumed, the intermediate is typically reacted in situ with a suitablereducing agent to provide the compounds of formula II. The overallconversion may be performed at about 0° C. to the boiling point of themixture, but room temperature is a preferred reaction temperature. Theformation of the compounds of formula II may take from 15 minutes to 24hours as measure by the consumption of the compound of formula (i).Methanol is typically a preferred solvent.

[0087] Suitable reducing agents include, but are not limited to,hydrogen over palladium or platinum on carbon, borane or complexes ofborane, e.g., borane-pyridine, borane-t-butylamine, andborane-dimethylamine complex; and borohydride reducing agents such assodium borohydride or sodium cyanoborohydride. Sodium cyanoborohydrideis a preferred reducing agent.

[0088] Compounds of formula (ii) and (iii) may be combined to preparedcompounds of formula III according to Scheme 4, wherein Y is definedsupra.

[0089] Compounds of formula (ii) may be converted to the correspondingacid halide by methods well known to one skilled in the art. Compoundsof formula III may be prepared by dissolving or suspending an acidhalide of a compound of formula (ii) in a suitable solvent and adding acompound of formula (iii) in a suitable solvent. Triethylamine ordimethylformamide is a suitable solvent and is typically preferred forthe compound of formula (ii). A 1:1 mixture of DMF and dichloromethaneis a convenient solvent and is typically preferred for the amine offormula (iii). This amide forming reaction is also preferably run in thepresence of 4-dimethylaminopyridine (DMAP).

[0090] The compound of formula (ii) is preferably employed in anequimolar amount, relative to the compound of formula (iii), but aslight excess (about a 0.05 to about 0.15 molar excess) is acceptable.DMAP is employed in a catalytic fashion. For example, about 5 molarpercent to about 15 molar percent, relative to the compound of formula(iii), is typically employed. A 10 molar percent is usually preferred.

[0091] Compounds of formula (iii), wherein Y is defined supra, are wellknown in the art and to the extent not commercially available, arereadily synthesized by standard procedures commonly employed in the art.

[0092] The synthesis of compounds of formula (ii) may be performed asdescribed in Route 1 below.

[0093] Compounds of formula XVI may be prepared by dissolving orsuspending a compound of formula XV and a suitable base in a suitablesolvent and adding a compound of formula XIV in a suitable solvent,dropwise. Toluene is a convenient solvent and is typically preferred.Triethylamine is the preferred base. The compound of formula XIV istypically and preferably employed in an equimolar amount, relative tothe compound of formula XV, but a slight excess is acceptable.

[0094] The reactants are preferably combined at about 0° C. and theresulting solution is typically warmed to room temperature and mixed forfrom about 18 hours to about 24 hours.

[0095] The compound of formula XVI may then be converted to the compoundof formula XIII by dissolving or suspending a compound of formula XVI ina suitable acidic solvent and adding hydroxylamine hydrochloride.Glacial acetic acid is a convenient acidic solvent and is typicallypreferred. The ester group is then hydrolyzed to the correspondingcarboxylic acid of formula (ii) through standard procedures commonlyemployed in the art, see for example, Larock, pgs 981-985.

[0096] The reactants are preferably combined at about room temperaturethen heated to reflux for from about 30 minutes to about 60 minutes.Preferably the reaction is heated to reflux from about 40 to 45 minutes.

[0097] Compounds of formula XIV and XV are known in the art and, to theextent not commercially available, are readily synthesized by standardprocedures commonly employed in the art.

[0098] Compounds of formula XIX may be prepared in a manner similar tothat described in the literature, for example, see Liu K, Shelton B R,Howe, R K, J. Org. Chem., 1980, 45, 3916-3918.

[0099] The pharmaceutical salts of the invention are typically formed byreacting a compound of formula I with an equimolar or excess amount ofacid or base. The reactants are generally combined in a mutual solventsuch as diethylether, tetrahydrofuran, methanol, ethanol, isopropanol,benzene, and the like for acid addition salts, or water, an alcohol or achlorinated solvent such as dichloromethane for base addition salts. Thesalts normally precipitate out of solution within about one hour toabout ten days and can be isolated by filtration or other conventionalmethods.

[0100] Acids commonly employed to form pharmaceutical acid additionsalts are inorganic acids such as hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid, phosphoric acid, and the like, andorganic acids such as p-toluenesulfonic, methanesulfonic acid,ethanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonicacid, succinic acid, citric acid, tartaric acid, benzoic acid, aceticacid, and the like. Preferred pharmaceutical acid addition salts arethose formed with mineral acids such as hydrochloric acid, hydrobromicacid, and sulfuric acid, and those formed with organic acids such asmaleic acid, tartaric acid, and methanesulfonic acid.

[0101] Bases commonly employed to form pharmaceutical base additionsalts are inorganic bases, such as ammonium or alkali or alkaline earthmetal hydroxides, carbonates, bicarbonates, and the like. Such basesuseful in preparing the salts of this invention thus include sodiumhydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate,sodium carbonate, sodium bicarbonate, potassium bicarbonate, calciumhydroxide, calcium carbonate, and the like. The potassium and sodiumsalt forms are particularly preferred.

[0102] It should be recognized that the particular counterion forming apart of any salt of this invention is not of a critical nature, so longas the salt as a whole is pharmacologically acceptable and as long asthe counterion does not contribute undesired qualities to the salt as awhole.

[0103] The optimal time for performing the reactions of the Schemes andthe Route can be determined by monitoring the progress of the reactionvia conventional chromatographic techniques. Furthermore, it ispreferred to conduct the reactions of the invention under an inertatmosphere, such as, for example, argon, or, particularly, nitrogen.Choice of solvent is generally not critical so long as the solventemployed is inert to the ongoing reaction and sufficiently solubilizesthe reactants to effect the desired reaction. The compounds arepreferably isolated and purified before their use in subsequentreactions. Some compounds may crystallize out of the reaction solutionduring their formation and then collected by filtration, or the reactionsolvent may be removed by extraction, evaporation, or decantation. Theintermediates and final products of formula I may be further purified,if desired by common techniques such as recrystallization orchromatography over solid supports such as silica gel or alumina.

[0104] The skilled artisan will appreciate that not all substituents arecompatible with all reaction conditions. These compounds may beprotected or modified at a convenient point in the synthesis by methodswell known in the art.

[0105] The following Preparations and Examples are provided to betterelucidate the practice of the present invention and should not beinterpreted in any way as to limit the scope of same. Those skilled inthe art will recognize that various modifications may be made while notdeparting from the spirit and scope of the invention. All publicationsmentioned in the specification are indicative of the level of thoseskilled in the art to which this invention pertains. The terms andabbreviations used in the instant Preparations and Examples have theirnormal meanings unless otherwise designated. For example “°C.”, “N”,“mmol”, “g”, “mL”, “M”, “HPLC”, “IR”, “MS(FD)”, “MS(IS)”, “MS(FIA)”,“MS(FAB)”, “MS(EI)”, “MS(ES)”, “UV”, and “¹H NMR”, refer to degreesCelsius, normal or normality, millimole or millimoles, gram or grams,milliliter or milliliters, molar or molarity, high performance liquidchromatography, infra red spectrometry, field desorption massspectrometry, ion spray mass spectrometry, flow injection analysis massspectrometry, fast atom bombardment mass spectrometry, electron impactmass spectrometry, electron spray mass spectrometry, ultravioletspectrometry, and proton nuclear magnetic resonance spectrometryrespectively. In addition, the absorption maxima listed for the IRspectra are only those of interest and not all of the maxima observed.

Preparation 1 3-Methyl-5-(2-chloro-6-fluorophenyl)4-isoxazolecarboxylicAcid Ethyl Ester

[0106] To a solution of ethyl 3-aminomethyl crotonate (4.79 g, 33.5mmol) in toluene (10 mL), was added triethylamine (3.73 g, 37 mmol). Thesolution was chilled using an ice bath, and then2-chloro-6-fluorobenzoyl chloride (6.47 g, 33.5 mmol) was added dropwiseover a 20 min period. The reaction was allowed to warm slowly to r.t.,and stirred for 24 hr. The resulting suspension was then filtered, andthe filtrate diluted with ethyl acetate (100 mL) and transferred to aseparatory funnel. The organic layer was sequentially washed with water,brine, dried (sodium sulfate), and the volatiles removed under reducedpressure to provide2-(2-chloro-6-fluorobenzoyl)-3-methylamino-but-2-enoic acid ethyl ester(9.46 g) as a golden solid, and primarily one geometrical isomer.

[0107] NMR (CDCl₃) δ 6.95-7.4 (3 m, 3H), 3.8 (m, 2H, OEt), 3.12 (d, 3H,—NCH₃), 2.4 (s, 3H, vinyl CH₃), 0.8 (t, OEt).

[0108] MS (ES) m/z 299.9 (M+H)⁺.

[0109] Crude adduct was then redissolved in glacial HOAc (50 mL) towhich was added NH₂OH.HCl (1.8 g, 1.1 eq). The solution was then heatedto reflux for 40-45 min to effect isoxazole formation. The reactionmixture was concentrated to an oil, diluted with ether, and transferredto a sep. funnel. The organic phase was washed with saturatedbicarbonate, brine, then dried. Filtration and concentration affordedcrude isoxazole ethyl ester (7.5 g), which could be used without furtherpurification.

[0110] MS (+ES) m/z 283.9 (M+H)⁺.

Preparation 2 4Isoxazolecarboxylic Acid, 3-Methyl,5-(2-chloro-6-fluorophenyl)

[0111] Hydrolysis of the ethyl ester was accomplished by dissolving thecrude ester (7.5 g, approx. 0.027 mol) in TBF (250 mL), and adding aq.LiOH (1.344 g in 100 mL, 2 eq). After stirring overnight at r.t., thesolution was concentrated to ⅔^(rd) volume, diluted with EtOAc (200 mL)and 50 mL water, transferred to a separatory funnel, and the aqueousphase collected. The organic phase was washed twice, and the combinedaqueous phase was then acidified with 5N HCl. Back extraction with threewashings of EtOAc was then followed with a brine wash of the combinedorganics. After drying over Na₂SO₄, filtration and concentration, cleanisoxazole acid was obtained (2.94 g).

[0112] MS (−ES) m/z 253.8, 255.8 (M−H)⁻.

Preparation 3 2-{3-[(T-butoxy)carbonylamino]cyclohexyl}acetic Acid

[0113]Phenylmethyl-2-{(3S,1R)-3-[(t-butoxy)carbonylamino]-cyclohexyl}acetate(1.0 g; 2.77 mmol) was dissolved in tetrahydrofuran (4 mL) and ethanol(4 mL) under a dry nitrogen atmosphere at room temperature. This cloudywhite solution became clear and colorless after mixing with 2NNaOH_((aq)) (15 mL; 19.4 mmol; 11.1 equiv) for 2 h. After rotaryevaporation to dryness, the white solid was dissolved in water (20 mL)and the resulting solution extracted with diethyl ether (twice).Acidification of the aqueous layer to pH 2 with 1N HCl_((aq)) produced awhite solid that was extracted into ethyl acetate (thrice). The ethylacetate was washed with saturated NaCl_((aq)), dried with Na₂SO_(4(s)),and concentrated to dryness by rotary evaporation. The resulting whitesolid (700 mg; 98% yield) was used in subsequent reactions withoutfurther purification.

[0114] MS(ES) calc'd: [M+Na]⁺=280.2 m/z, [M−H]⁻=256.2 m/z. Found: 280.1m/z; 256.2 m/z.

Preparation 4 N-((1S,3R)-3-{[(phenylmethoxy)carbonylamino]methyl}Cyclohexyl)(t-butoxy)carboxamide

[0115] To a solution of 2-{3-[(t-butoxy)carbonylamino]cyclohexyl}aceticacid (3.43 g, 13.35 mmol), Et₃N (3.75 mL, 26.96 mmol) in toluene (86 mL)under N₂ was added DPPA (5.8 mL, 26.96 mmol) and benzyl alcohol (4.28mL, 41.38 mmol). The solution was heated to reflux overnight. Thereaction was cooled to room temperature, diluted with EtOAc, washed(1.0N NaOH then brine), dried (MgSO₄), filtered, and concentrated.Column chromatography (silica gel, hexanes/EtOAc gradient) gave thetitle compound (3.05 g, 63%). Mass Spectrum (ES+) (m/z) 263.1 [M-BOC].

Preparation 5 (cis-3(S)-Aminocyclohexylmethyl)carbamic Acid Benzyl Ester

[0116] S-amino enantiomer of a compound from preparation 4 (1.0 g, 2.76mmol) was treated with TFA (5 mL) under N₂. After 20 min of stirring atr.t. the reaction was complete. The crude was then concentrated to anoil which was purified on a Varian Bond-Elut SCX column (10 g). Thecolumn was eluted consecutively with CHCl₃, MeOH, and ammonia (2.0M inMeOH). The pure product was recovered from the ammonia fractions. Thesolvent was removed in vacuo to afford 0.632 g (87%) as a colorless oil.MS (ES+) m/z 263.0 (M+H)⁺.

Preparation 6(1S,3R)(3-{[5-(2-Chloro-6-fluorophenyl)-3-methylisoxazole-4-carbonyl]amino}cyclohexylmethyl)carbamicAcid Benzyl Ester

[0117] A solution from preparation 2 (1.0 g, 3.9 mmol) in toluene (30mL) was treated with a catalytic amount of pyridine (0.1 mL) and cooledto 0° C. The solution was then treated with oxalyl chloride (0.545 g,4.3 mmol) and stirred at r.t. for 2 hr. 1H NM showed the completion ofthe acid chloride formation; 7.52 (d, 1H), 7.37 (d, 1H), 7.15 (t, 1H).The solvent was removed in vacuo. A solution from preparation 5 (0.550g, 2.09 mmol) and triethylamine (0.422 g, 4.18 mmol) in dry DMF (25 mL)was stirred at r.t. This solution was then treated with the acidchloride from above (0.859 g, 3.135 mmol) which was added dropwise overtwo min. The reaction was then catalyzed with DMAP (0.025 g, 0.21 mmol)and allowed to stir o.n. The reaction was diluted in EtOAc (250 mL),transferred to a sep funnel, and washed with a 5% citric acid solution(5×50 mL), sat. sodium bicarbonate solution (2×50 mL), brine (2×50 mL)and was dried over sodium sulfate. The EtOAc solution was filtered andthe solvent removed in vacuo to yield an orange solid. The solid waspurified using silica gel column chromatography. The column was preparedwith CHCl₃ and the product was eluted with 10% EtOAc in CHCl₃. Thesolvent was removed in vacuo to afford 0.584 g (56%) of the titlecompound as a white solid. MS (ES+) m/z 500.1 (M+H)⁺.

Preparation 7(1R,3S)-[3-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,5-c]quinolin-5-yl)-cyclohexylmethyl]-carbamicAcid Benzyl Ester

[0118] A solution of the product from preparation 6 (0.534 g, 1.1 mmol)in dry DMF (20 mL) was stirred and cooled to 0° C. in an ice bath. Thereaction was then treated with KHMDS-0.5M in toluene (4.300 mL, 2.140mmol) dropwise over 5 min and allowed to warm back to r.t. The reactionwas complete in 25 min. The reaction mixture was then diluted in EtOAc(200 mL), transferred to a sep funnel, and washed consecutively with 5%citric acid solution (4×50 mL), sat sodium bicarbonate (2×50 mL), brine(2×50 mL), and dried over sodium sulfate. The solvent was removed invacuo to afford a yellow-white solid. The solid was purified usingsilica gel column chromatography and an eluting solvent of 100% CHCl₃.The solvent was removed in vacuo to afford 0.401 g (78%) as a whitesolid. MS (ES+) m/z 480.1 (M+H)⁺.

Preparation 8(1R,3S)-5-(3-Aminomethyl-cyclohexyl)-9-chloro-3-methyl-5H-isoxazolo[4,5-c]quinolin-4-one

[0119] A solution of the product from preparation 7 (0.40 g, 0.83 mmol)in dry CH₂Cl₂ (5 mL) was treated with iodotrimethylsilane (0.25 g, 1.25mmol) and stirred at r.t. After 3 hr the reaction was quenched with MeOH(2 mL) and stirred for an additional 30 min. The solution was thenconcentrated to an orange solid and taken up in EtOAc (20 mL) and 1 NHCl (20 mL) and transferred to a sep funnel. The organic layer wasextracted and the aqueous layer was washed with additional EtOAc (2×10mL). The pH of the aqueous layer was then adjusted to ph˜12 with 5 MNaOH. The product was extracted with EtOAc (3×25 mL). The EtOAcextractions were dried over sodium sulfate, filtered, and the solventremoved in vacuo to afford 0.240 g (84%) as a white solid which was usedwithout further purification. MS (ES+) m/z 346.0 (M+H)⁺.

Preparation 9(1R,3S)-N-[3-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,5-c]quinolin-5-yl)-cyclohexylmethyl]-nicotinamide

[0120] A solution of the product from preparation 8 (0.342 g, 0.99 mmol)and nicotinoyl chloride (0.210 g, 1.49 mmol) in CH₂Cl₂ (25 mL) wastreated with triethylamine (0.1 g, 0.99 mmol) and stirred o.n. at r.t.The reaction was concentrated to a solid and diluted in EtOAc (50 mL),washed with a sat. bicarbonate solution (3×25 mL), brine (2×25 mL), anddried over sodium sulfate. The solvent was removed in vacuo to afford ayellow oil. The oil was purified by silica gel column chromatography.The column was prepared using 100% EtOAc and the product was eluted offthe column with 5% MeOH in EtOAc. The solvent was removed in vacuo toafford 0.185 g (41%) as a white solid. MS (ES+) m/z 451.0 (M+H)⁺, (ES−)m/z 449.0 (M−H)⁻, 509.1 (M+CH₃COO⁻)⁻.

Preparation 10N-[3-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,5-c]quinolin-5-yl)-cyclohexylmethyl]-6-fluoro-nicotinamide

[0121] 6-Fluoro-nicotinic acid (0.05 g, 0.35 mmol) and oxalyl chloride(0.055 g, 0.44 mmol) were combined in toluene (10 mL) and stirred for 2hr at 80° C. to form the acid chloride. The reaction was concentrated toa solid and added directly to a solution of a compound from preparation9 (0.1 g, 0.29 mmol) in CH₂Cl₂ (15 mL). The reaction was treated withexcess Et₃N (0.06 g, 0.58 mmol) and stirred at room temperatureovernight. The reaction was then diluted in CH₂Cl₂ (85 mL) and washedwith a saturated sodium bicarbonate solution (3×50 mL) and brine (2×50mL). The organic solution was dried over sodium sulfate, filtered, andthe solvent removed to afford a crude yellow solid. The solid waspurified on a chromatotron using a 1 cm thick silica gel plate. Theproduct was eluted with 50% EtOAc in CH₂Cl₂. The solvent was removed toafford 0.084 g (61%) of the title compound as a white solid. MS m/z(ES+) 468.8 (M+H)⁺, (ES−) 466.8 (M−H)⁻, 526.8 (M+CH3COO⁻)⁻.

Preparation 11(1R,3S)-N-[3-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,5-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide

[0122] A solution of the product from preparation 8 (0.240 g, 0.696mmol) and benzoyl chloride (0.147 g, 1.044 mmol) in CH₂Cl₂ (5 mL) wastreated with triethylamine (0.07 g, 0.696 mmol) and stirred o.n. at r.t.The reaction was diluted in CH₂Cl₂ (25 mL), washed with a 5% citric acidsolution (2×10 mL), brine (2×10 mL), and dried over sodium sulfate. Thesolvent was removed in vacuo to afford a yellow oil. The oil waspurified by silica gel column chromatography. The column was preparedusing 2:1 Hexanes:CH₂Cl₂ and the product was eluted with 5% MeOH inCH₂Cl₂. The solvent was removed in vacuo to afford 0.300 g (97%) as awhite solid. MS (ES+) m/z 500.1 (M+H)⁺.

EXAMPLE 1{3-[3-(1-Amino-ethylidene)-5-chloro-2,4-dioxo-3,4-dihydro-2H-quinolin-1-yl]-cyclohexylmethyl}-carbamicAcid Benzyl Ester

[0123] The compound of preparation 7 (0.4 g, 0.8 mmol) and Mo(CO)₆ (0.44g, 1.7 mmol) were combined in a solution of acetonitrile (25 mL) andwater (5 mL). The reaction mixture was heated to reflux while stirring.After stirring for 3 hr the reaction was complete. The reaction wasconcentrated to a dark brown solid under vacuum. The solid was dilutedin CH₂Cl₂ (10 mL) and was purified by silica gel column chromatographyusing 50% EtOAc in CH₂Cl₂ to elute the product. The solvent was removedin vacuo to afford 0.325 g of desired product as a light yellow oil. MS(ES+) m/z 481.9 (M+H)⁺, (ES−) 479.9 (M−H)⁻.

EXAMPLE 2N-{3-[3-(1-Amino-ethylidene)-5-chloro-2,4-dioxo-3,4-dihydro-2H-quinolin-1-yl]-cyclohexylmethyl}-nicotinamide

[0124] The compound of preparation 9 (0.02 g, 0.04 mmol) and Mo(CO)₆(0.13 g, 0.5 mmol) were combined in a solution of acetonitrile (5 mL)and water (1 mL). The reaction mixture was heated to 60° C. and stirredfor 2 hr. The reaction was concentrated to a dark brown solid undervacuum. The solid was diluted in CH₂Cl₂ (10 mL) and purified by passingdirectly through a Bond-Elut cation exchange column. The product waseluted with 2M Ammonia in MeOH. The brown liquid was filtered using aGelman Nylon Acrodisc to afford a yellow solution. The solvent wasremoved in vacuo to afford 0.018 g (90%) of a white solid. MS (ES+) m/z452.9 (M+H)⁺, (ES−) m/z 450.8 (M−H)⁻, 510.9 (M+CH₃COO⁻)⁻.

EXAMPLE 3N-{3-[3-(1-Amino-ethylidene)-5-chloro-2,4-dioxo-3,4-dihydro-2H-quinolin-1-yl]-cyclohexylmethyl}-6-fluoro-nicotinamide

[0125] A compound from preparation 10 (0.05 g, 0.11 mmol) and Mo(CO)₆(0.31 g, 1.2 mmol) were combined in a solution of acetonitrile (15 mL)and water (3 mL). The reaction mixture was heated to 60° C. and stirredfor 2 hr. The reaction was concentrated to a dark brown solid undervacuum. The solid was diluted in CH₂Cl₂ (10 mL) and purified by silicagel column chromatography using 50% EtOAc in CH₂Cl₂ to elute the desiredproduct. The solvent was removed in vacuo to afford 0.022 g (44%) of awhite solid. MS (ES+) m/z 470.9 (M+H)⁺, (ES−) m/z 468.8 (M−H)⁻.

EXAMPLE 4N-{3-[3-(1-Amino-ethylidene)-5-chloro-2,4-dioxo-3,4-dihydro-2H-quinolin-1-yl]-cyclohexylmethyl}-benzamide

[0126] The compound of preparation 11(0.05 g, 0.1 mmol) and Mo(CO)₆ (0.3g, 1.1 mmol) were combined in a solution of acetonitrile (5 mL) andwater (1 mL)*. The reaction mixture was heated to 60° C. while stirring.After 3 hr of stirring the reaction was complete. The reaction wasconcentrated to a dark brown solid under vacuum. The solid was dilutedin CH₂Cl₂ (1 mL) and purified by passing through a Varian Bond Elut SIcolumn (5 g). The product was eluted with 2% MeOH in CH₂Cl₂. The solventwas removed in vacuo to afford 0.041 g of the title compound as a lightbrown solid, which was used as is in following experiments. MS (ES+) m/z452.0 (M+H)⁺, (ES−) 450.0 (M−H)⁻.

[0127] The compounds of the invention are inhibitors of MRP1. Thus, thecompounds of the invention may be used to inhibit any neoplasm havingintrinsic and/or acquired resistance, conferred in part or in total byMRP1, to an oncolytic or oncolytics. In other words, treatment of such aneoplasm with an effective amount of a compound of this invention willcause the neoplasm to be more sensitive to chemotherapy that wasrendered less efficacious by MRP1.

[0128] Vincristine, epirubicin, daunorubicin, doxorubicin, and etoposideare oncolytics that are substrates of MRP1. See Cole, et. al.,“Pharmacological Characterization of Multidrug Resistant MRP-transfectedHuman Tumor Cells”, Cancer Research, 54:5902-5910, 1994. Since MRP1 isubiquitous in mammals, particularly humans, Nooter, K, et. al.,“Expression of the Multidrug Resistance-Associated Protein (MRP) Gene inHuman Cancers”, Clin. Can. Res., 1:1301-1310, (1995), chemotherapy whosegoal is to inhibit a neoplasm employing any of those agents has thepotential to be rendered less efficacious by MRP1. Thus, neoplasms ofthe bladder, bone, breast, lung (small-cell), testis, and thyroid andmore specific types of cancer such as acute lymphoblastic andmyeloblastic leukemia, Wilm's tumor, neuroblastoma, soft tissue sarcoma,Hodgkin's and non-Hodgkin's lymphomas, and bronchogenic carcinoma may beinhibited with a combination of one or more of the above oncolytics anda compound of this invention.

[0129] The biological activity of the compounds of the present inventionwas evaluated employing an initial screening assay, which rapidly andaccurately measured the activity of the tested compound in inhibitingMRP1 or MDR1. Assays useful for evaluating this reversing capability arewell known in the art. See, e.g., T. McGrath, et al., BiochemicalPharmacology, 38:3611, 1989; D. Marquardt and M. S. Center, CancerResearch, 52:3157, 1992; D. Marquardt, et al., Cancer Research, 50:1426,1990; and Cole, et. al., Cancer Research, 54: 5902-5910, 1994.

[0130] Assay for Reversal of MRP1-Mediated Doxorubicin Resistance andMDR1-Mediated Vincristine Resistance: HL60/Adr and HL60/Vinc arecontinuous cell lines, which were selected for doxorubicin andvincristine resistance respectively by culturing HL60, a human acutemyeloblastic leukemia cell line, in increasing concentrations ofdoxorubicin or vincristine until a highly resistant variant wasattained.

[0131] HL60/Adr and HL60/Vinc cells were grown in RPMI 1640 (Gibco)containing 10% fetal bovine serum (FBS) and 50 μg/ml GENTAMICIN™(Sigma). Cells were harvested; washed twice with assay medium (same asculture media); counted; and diluted to 1×10⁵ cells/ml in assay medium.One hundred microliters of cells were aliquoted into wells of a 96 welltissue culture plate. Two columns of each 96 well plate served as anegative control and received assay medium containing no cells.

[0132] Test compounds and reference compounds were dissolved in dimethylsulfoxide (DMSO) at a concentration of 5 mM. Samples were diluted inassay medium and 25 μl of each test compound was added to 8 wells. Assaystandards were run in quadruplicate. Assay media was added to half ofthe wells and doxorubicin to the other half of the wells to achieve afinal volume of 150 μl per well.

[0133] The plates were incubated at 37° C. for 72 hours in a humidifiedincubator with a 5% carbon dioxide atmosphere. Cell viability andvitality was measured by oxidation of a alamarBlue™ fluorescent dyeusing standard conditions. The plates were incubated for 3 hours at 37°C. Fluorescence was determined using 550 nm excitation and 590 nmemission using a microtitre plate reader.

[0134] The ability of a test compound to reverse the resistance ofHL60/Adr and HL60/Vinc cells to doxorubicin was determined by comparisonof the absorbance of the wells containing a test compound in addition tothe oncolytic (doxorubicin) with the absorbance of wells containing theoncolytic without a test compound. Controls were used to eliminatebackground and to ensure the results were not artifactual. The resultsof the assay are expressed as percent inhibition of cell growth. Theoncolytic alone at the tested concentration minimally inhibits thegrowth of HL60/Adr or HL60/Vinc cells.

[0135] Representative compounds of formula I demonstrated a significanteffect in reversing the MRP1 multiple drug resistance. Many of thecompounds showed very significant enhancement of activity in combinationwith the oncolytic agent as opposed to the oncolytic agent alone. Inaddition, a large majority of the compounds tested displayed asignificant degree of selective inhibition of the HL60/Adr cell lineover the HL60/Vinc cell line.

[0136] When administering an oncolytic in practicing the methods of thisinvention, the amount of oncolytic employed will be variable. It shouldbe understood that the amount of the oncolytic actually administeredwill be determined by a physician, in the light of the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the actual oncolytic administered, the age, weight,and response of the individual patient (mammal), and the severity of thepatient's symptoms. Of course, the amount of oncolytic administeredshould be decided and closely monitored by that patient's physician.After deciding on the oncolytic or oncolytics to employ, “ThePhysician's Desk Reference®”, published by Medical Economics Company atMontvale, N.J. 07645-1742, is a helpful resource to the physician indeciding on amounts of the oncolytic to administer and is updatedannually.

[0137] Preferred formulations, and the methods of this inventionemploying those formulations, are those which do not contain anoncolytic. Thus, it is preferred to administer the compounds of thisinvention separately from the oncolytic. The oncolytics mentioned inthis specification are commercially available and may be purchased inpreformulated forms suitable for the methods of this invention.

[0138] The compounds of formula I alone, or optionally in combinationwith an oncolytic, are usually administered in the form ofpharmaceutical formulations. These formulations can be administered by avariety of routes including oral, rectal, transdermal, subcutaneous,intravenous, intramuscular, and intranasal. Such formulations areprepared in a manner well known in the pharmaceutical art and compriseat least one active compound of formula I.

[0139] The present invention also includes methods employingpharmaceutical formulations, which contain, as the active ingredient,the compounds of formula I, and optionally an oncolytic, associated withpharmaceutical carriers. In making the formulations of the presentinvention the active ingredient(s) is usually mixed with an excipient,diluted by an excipient, or enclosed within such a carrier which can bein the form of a capsule, sachet, paper or other container. When theexcipient serves as a diluent, it can be a solid, semi-solid, or liquidmaterial, which acts as a vehicle, carrier or medium for the activeingredient. Thus, the formulations can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing for example up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

[0140] In preparing a formulation, it may be necessary to mill theactive compound(s) to provide the appropriate particle size prior tocombining with the other ingredients. If the active compound(s) issubstantially insoluble, it ordinarily is milled to a particle size ofless than 200 mesh. If the active compound(s) is substantially watersoluble, the particle size is normally adjusted by milling to provide asubstantially uniform distribution in the formulation, e.g., about 40mesh.

[0141] Some examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxybenzoates; sweetening agents; and flavoring agents. Theformulations of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

[0142] The formulations are preferably formulated in a unit dosage form,each dosage containing from about 5 to about 100 mg, more usually about10 to about 30 mg, of each active ingredient. The term “unit dosageform” refers to physically discrete units suitable as unitary dosagesfor human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

[0143] The compounds of formula I are effective over a wide dosagerange. For example, dosages per day normally fall within the range ofabout 0.5 to about 30 mg/kg of body weight. In the treatment of adulthumans, the range of about 1 to about 15 mg/kg/day, in single or divideddose, is especially preferred. However, it will be understood that theamount of the compound actually administered will be determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound administered, the age, weight, and response of the individualpatient, and the severity of the patient's symptoms, and therefore theabove dosage ranges are not intended to limit the scope of the inventionin any way. In some instances dosage levels below the lower limit of theaforesaid range may be more than adequate, while in other cases stilllarger doses may be employed without causing any harmful side effect,provided that such larger doses are first divided into several smallerdoses for administration throughout the day.

[0144] For preparing solid formulations such as tablets the principalactive ingredient(s) is mixed with a pharmaceutical excipient to form asolid preformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient(s) is dispersed evenly throughout the formulation so that theformulation may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention.

[0145] The tablets or pills of the present invention may be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by entericlayer, which serves to resist disintegration in the stomach and permitthe inner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

[0146] The novel formulations which are liquid forms may be incorporatedfor administration orally or by injection and include aqueous solutions,suitably flavored syrups, aqueous or oil suspensions, and flavoredemulsions with edible oils such as cottonseed oil, sesame oil, coconutoil, or peanut oil, as well as elixirs and similar pharmaceuticalvehicles.

[0147] Formulations for inhalation or insufflation include solutions andsuspensions in pharmaceutical, aqueous or organic solvents, or mixturesthereof, and powders. The liquid or solid formulations may containsuitable pharmaceutical excipients as described supra. Preferably theformulations are administered by the oral or nasal respiratory route forlocal or systemic effect. Compositions in preferably pharmaceuticalsolvents may be nebulized by use of inert gases. Nebulized solutions maybe breathed directly from the nebulizing device or the nebulizing devicemay be attached to a face mask, tent, or intermittent positive pressurebreathing machine. Solution, suspension, or powder formulations may beadministered, preferably orally or nasally, from devices, which deliverthe formulation in an appropriate manner.

We claim:
 1. A compound of formula I:

wherein: E is a bond or —C(R⁴)(R⁴)—; R¹ is independently at eachoccurrence hydrogen or C₁-C₆ alkyl; R² is independently at eachoccurrence hydrogen or C₁-C₆ alkyl; R³ is independently at eachoccurrence hydrogen, C₁-C₆ alkyl, optionally substituted C₃-C₈cycloalkyl, optionally substituted (C₁-C₄ alkyl) C₃-C₈ cycloalkyl,optionally substituted (C₁-C₄ alkyl)-aryl, optionally substituted aryl,optionally substituted (C₁-C₄ alkyl)-heterocycle, optionally substitutedheterocycle, C₁-C₆ alkoxy, optionally substituted O—(C₃-C₈ cycloalkyl),optionally substituted (C₁-C₄ alkoxy) C₃-C₈ cycloalkyl, optionallysubstituted (C₁-C₄ alkoxy)-aryl, optionally substituted O-aryl,optionally substituted (C₁-C₄ alkoxy)-heterocycle, or optionallysubstituted O-heterocycle; R⁴ is independently at each occurrencehydrogen or C₁-C₆ alkyl; R⁵ is independently at each occurrence hydrogenor C₁-C₆ alkyl; or a pharmaceutical salt thereof.
 2. A method ofinhibiting MRP1 in a mammal which comprises administering to a mammal inneed thereof an effective amount of a compound of formula I.
 3. A methodof inhibiting a resistant neoplasm, or a neoplasm susceptible toresistance in a mammal which comprises administering to a mammal in needthereof an effective amount of a compound of formula I in combinationwith an effective amount of an oncolytic agent.
 4. A pharmaceuticalformulation comprising a compound of formula I in combination with oneor more oncolytics, pharmaceutical carriers, diluents, or excipientstherefor.
 5. A pharmaceutical formulation comprising a compound offormula I.